Abstract: Mechanical forces exerted by cells control processes of central importance in modern biology and medicine, for example cancer metastasis, stem cell differentiation, and embryonic development. However, the mechanisms by which cells exert and detect force remain poorly understood. Our understanding of how mechanical signaling modulates the behavior of whole tissues or organs is likewise in its infancy. We will use the tools of single-molecule biophysics to test transformative hypotheses about the roles of mechanical force in biology. Current techniques do not provide quantitative measurements of forces within and between cells. We will use novel molecular force sensors to directly observe myosin-based tension generation in living cells. These measurements will provide an unprecedented look at cells as mechanical entities-we will watch cells exert and respond to force in real time. Our measurements thus constitute a radical departure from the traditional forms of microscopy that characterize cellular structure, but that are blind to the underlying mechanical forces that shape and maintain both cells and tissues. Our work will clarify long-standing controversies about how the cytoskeleton is constructed, with important implications for our understanding of stem cell differentiation and cancer metastasis. In a separate set of measurements we will test the hypothesis that mechanical forces directly modulate the remodeling of the extracellular matrix by matrix metalloproteinases (MMPs). Confirmation of this model will open up new avenues in the investigation of heart disease. Further, our measurements will provide crucial insight into the mechanism by which MMPs differentially recognize substrates, thus contributing to the development of improved treatments for cancer. Finally, we will integrate these two strands of inquiry by measuring both myosin force generation and extracellular matrix remodeling in whole Drosophila embryos. Our experiments in Drosophila represent a first step toward a quantitative understanding of molecular force generation and mechanical signaling in living organisms. We feel that this transition to in vivo measurement represents a necessary progression both in our own research and for the field of biophysics as a whole. Public Health Relevance: Cells use nanometer-sized molecular motors to move, grow, and divide. Cells inside the human body also pull and tug on each other. This mechanical signaling is a crucial component of normal growth and development, but failures in mechanical communication can result in to the development of multiple diseases. We will watch cells in living organisms create and respond to force in real time. By learning more about how cellular mechanical signaling works we will be better able to understand and treat cancer, heart disease, and other important illnesses.